Formation of a functional, multi-chambered heart involves a complex regulatory network of signaling molecules and transcription factors. Perturbations at any step in this process often result in severe morphological and/or functional defects. Thus, understanding the function of cardiac developmental control genes will reveal important clues into the genetic basis of congenital heart malformations. A transcription factor that plays an important role in cardiac muscle development is the MEF2 protein family. Loss-of function mutations in rnef2 in flies and in mice disrupt the appropriate development of cardiac muscle. Hence, a comprehensive in vivo analysis of mef2 will lead to a better understanding of the genetics and molecular mechanisms of heart development. In vertebrates, MEF2 is encoded by four genes, mef2a,-b,-c, and -d that are coexpressed in muscle and non-muscle lineages and whose functions in vitro are largely interchangeable. However, targeted mutations in mice have revealed distinct roles for mef2a and mef2c in cardiac development, mef2a null mice exhibit a post-natal cardiomyopathy with severe cytoarchitectural and mitochondrial defects in the multi-chambered heart whereas mef2c mutant mice display altered cardiac looping morphogenesis during embryogenesis. Given the distinct cardiac phenotypes, the mechanisms by which closely related genes within a multigene family uniquely regulate cellular differentiation in vivo will be explored. Because the mef2a knockout mice represent a potential connection to human cardiac disease a thorough developmental and genetic analysis will be initiated. The proposed research will address these questions using gene targeting and transgenic approaches in mice. The specific aims are: 1) to generate a conditional knockout for the mef2a gene to determine the tissue-specific and temporal requirement for mef2a in cardiac muscle cells and the role of mef2a in the adult heart, 2) to create knock-in alleles for the mef2a and mef2c genes to examine possible functional redundancy between these two vertebrate mef2 genes, and 3) to dissect the molecular mechanisms of the post-natal cardiomyopathy in mef2a knockout mice by identifying and characterizing genes dysregulated in mutant hearts. The elucidation of MEF2-dependent transcriptional pathways in vivo will be an important step in the development of genetic strategies to treat cardiovascular disease.